Process for culturing cells sampled for biopsy

ABSTRACT

The present invention provide: a novel process for culturing animal cells and a kit for culturing animal cells, in which, even if the number of cells as sampled for biopsy is extremely small, the proliferation can sufficiently be maintained so as to enable to carry out various culture and/or tests, especially anticancer agent sensitivity tests, and the contamination with bacteria can be inhibited without damaging physiological activity of cells, especially sensitivity to anticancer agents. The process for culturing animal cells, according to the present invention, comprises the step of culturing a sample containing animal cells obtained from living body tissue in order to subject the sample to further culture and/or a test, with the process being characterized in that a culture medium is used wherein the culture medium has a proliferating action and physiological activity-retaining action on the animal cells, and further has a killing action and/or multiplication-inhibition action on bacteria.

BACKGROUND OF THE INVENTION

A. Technical Field

The present invention relates to a process for culturing animal cellsobtained from living body tissue. More particularly, the inventionrelates to a process for culturing animal cells obtained from livingbody tissue in order to use them for further culture and/or a test.

B. Background Art

Anticancer agent sensitivity tests sometimes have been carried out usingsubcultured cancer cells hitherto, but for the purpose of evaluatingeffects of anticancer agents upon respective individuals, methods ofevaluating the effects of the anticancer agents by utilizing so-calledprimary culture, in which samples taken from a living body are directlycultured, are widely employed. However, when cancer cells were takenfrom living body tissue as a sample and then used, there were problemssuch that: a necessary and sufficient amount of live cells maintainingproliferativity cannot be obtained, and in addition, the sample containsnormal cells other than the aimed cancer cells, and further containsmany other components such as bacteria.

Accordingly, as a method for efficiently carrying out anticancer agentsensitivity tests, for example, the following method has been applied inrecent year: a method in which primary cells containing cancer cells ina collagen gel is embedded, and then cultured. Even if the absoluteamount of the cancer cells as used for the tests is small, the cancercells can favorably be proliferated in the collagen gel. In addition,even if the collagen gel is contaminated with cells other than thecancer cells such as fibroblast cells and each is grown and extended,they can easily be distinguished morphologically. Therefore, theevaluation of the sensitivity of only the cancer cells both theanticancer agents are added thereto and not can easily be carried out.

Furthermore, for the purpose of enhancing the accuracy of the tests,well known is a method that involves: placing a droplet of acancer-cell-containing collagen solution on a surface of a supportingbase material; allowing the droplet to gel; and forming and embedding aglobular collagen gel, in order to culture the cancer cells under morefavorable density and circumstances to proliferated the cancer cellsmore easily (Japanese Patent No. 2879978).

These methods may be theoretically and technically preferred, but, whensuch as anticancer agent sensitivity tests are carried out using cellssampled for biopsy as actually sampled from living bodies, the testsuccess rate is still low, and the level of the tests is not sufficientto say that it is in on a realistic and practical level in the clinical.As to its cause, the following is mentioned first: there are many caseswhere cells of which the number has a level such that the test cansufficiently be carried out cannot be collected, or the cells cannot becollected at all. In order to solve this, it is thought necessary toreduce the loss of the number of the cells as sampled from the livingbodies as low as possible. However, the washing step regarded asnecessary in conventional methods has problems such that: it is verydifficult to carry out the step for a very slight sample amount ofbiopsy materials; the sufficient washing is impossible; and further theyield is very low and the loss is increased. As to the second cause, thefollowing is mentioned: the culture cannot be continued often because ofthe contamination with such as bacteria. It is natural to cause thepossibility of the contamination on the operation, and tissue sampled invivo especially from such as intestines includes Escherichia coli andvarious bacteria that are bacteria ordinary living in vivo. Therefore,under the present circumstances, even if the process of theabove-mentioned washing step is gone through, the inhibition of thecontamination still cannot entirely be performed, resulting in seriousproblems.

SUMMARY OF THE INVENTION A. Object of the Invention

Accordingly, an object of the present invention is to provide: a novelprocess for culturing animal cells; and a kit for culturing animalcells; by which: even if the number of cells as sampled for biopsy isextremely small, the proliferation can be maintained sufficiently toenable the implementation of such as various culture and/or tests,especially anticancer agent sensitivity tests, and the contaminationwith bacteria can be inhibited without damaging physiological activity,especially, sensitivity to anticancer agents, of cells.

B. Disclosure of the Invention

The present inventors diligently studied to solve the above-mentionedproblems. As a result, when they culture and proliferate cells sampledfor biopsy in order to use them for further culture and/or a test, theytook note of a culture medium as used during the culture, and then theyfound that: if a culture medium having specific action and effect isused as this culture medium, all the above-mentioned problems can oncebe solved.

That is to say, a process for culturing animal cells under embeddedconditions, according to the present invention, comprises the step ofculturing sample cells containing animal cells obtained from living bodytissue in order to subject the sample cells to further culture and/or atest, with the process being characterized in that a culture medium isused wherein the culture medium has a proliferating action andphysiological activity-retaining action on the animal cells, and furtherhas a killing action and/or multiplication-inhibition action onbacteria.

In addition, a kit for culturing animal cells, according to the presentinvention, is a kit that comprises the following constituent articles: acollagen solution; a concentrated culture medium; a reconstitutingbuffer solution; an enzyme for dispersing cells; a tube for a culturesupporting base; a culture medium for preliminary culture; a serum-freeculture medium; and a cell-dyeing agent.

These and other objects and the advantages of the present invention willbe more fully apparent from the following detailed disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the cellproliferativity in the preliminary culture step (concerning a cancercell line and a fibroblast cell line).

FIG. 2 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the cellproliferativity in the preliminary culture step (concerning primary lungcancer cells).

FIG. 3 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the cellproliferativity in the proliferative culture step (concerning a cancercell line and primary lung cancer cell).

FIG. 4 is a graph representing an influence of the vancomycinconcentration in the preliminary culture upon step the cellproliferativity in the proliferative culture step (concerning afibroblast cell line).

FIG. 5 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the anticancer agentsensitivity test results (concerning a human lung cancer cell lineA-549).

FIG. 6 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the anticancer agentsensitivity test results (concerning a human gastric cancer cell lineMKN-28).

FIG. 7 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the anticancer agentsensitivity test results (concerning a human colon cancer cell lineC-1).

FIG. 8 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the anticancer agentsensitivity test results (concerning a human fibroblast cell lineHFL-1).

FIG. 9 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the anticancer agentsensitivity test results (concerning a human fibroblast cell lineWI-38).

FIG. 10 is a graph representing an influence of the vancomycinconcentration in the preliminary culture step upon the anticancer agentsensitivity test results (concerning human primary lung cancer cellsLung-k).

FIG. 11 is a graph representing an influence of the surface area of thesupporting base, as used in the preliminary culture step, upon theinitial adhesion of the tumor cells.

FIG. 12 is a graph representing an influence of the surface area of thesupporting base, as used in the preliminary culture step, upon the cellproliferativity ratio of the tumor cells.

FIG. 13 is a partial perspective sectional view of a receptacle that isone example of the culture supporting base that can be used for theprocess for culturing animal cells according to the present invention.

FIG. 14 is a photo of three containers as stood in a row wherein thecontainer is one example of the culture supporting base that can be usedfor the process for culturing animal cells according to the presentinvention.

FIG. 15 is a graph concerning the effect (in terms of OD₅₄₀) of theshape of the supporting base, as used in the preliminary culture step,upon collection of living cells of tumor cells.

FIG. 16 is a graph concerning the effect (in terms of the number ofliving cells) of the shape of the supporting base, as used in thepreliminary culture step, upon collection living cells of tumor cells.

FIG. 17 is a graph concerning the effect (in terms of OD₅₄₀) of theshape of the supporting base, as used in the preliminary culture step,upon collection of living cells of tumor cells.

FIG. 18 is a graph concerning the effect (in terms of the number ofliving cells) of the shape of the supporting base, as used in thepreliminary culture step, upon collection living cells of tumor cells.

FIG. 19 is a graph representing the cell proliferativity ratio of cellsduring the embedding culture wherein the cells are derived from cultureon each supporting base.

FIG. 20 is a graph representing the cell proliferativity ratio of cellsduring the embedding culture wherein the cells are derived from cultureon each supporting base.

DETAILED DESCRIPTION OF THE INVENTION

The process for culturing animal cells, according to the presentinvention, comprises the step of culturing sample cells containinganimal cells obtained from living body tissue in order to subject thesample cells to further culture and/or a test, with the process beingcharacterized in that a culture medium is used wherein the culturemedium has a proliferating action and physiological activity-retainingaction on the animal cells, and further has a killing action and/ormultiplication-inhibition action on bacteria.

Herein, in the present invention, it is favorable that the samplecontaining animal cells obtained from living body tissue is usually asection of internal organs or tissue (living body tissue) separated froma living body. Such an animal cell obtained from living body tissue,that is, a cell as sampled for biopsy, is called a primary cell and usedfor what is called primary culture. In addition, typical examples of theprimary cell include a tumor cell. Of this tumor cell, a malignant tumorcell, that is, a cancer cell is an object particularly when theanticancer agent sensitivity tests are carried out. The samplecontaining animal cells obtained from living body tissue, that is, aculture sample, most preferably consists of cells which are objects ofculture (e.g. cancer cells) alone, but usually it also contains normalcells such as fibroblast cells as a portion of living body tissue. Inaddition, it often occurs that dead cells, in addition to live cells,are contained as the aimed cells themselves.

In addition, subjecting the sample to further culture and/or a test inthe present invention may mean using at least one portion or theentirety of a sample cultured by the present invention culturing processas a sample for desired culture to be carried out newly andcontinuously, or using it as a sample which is a direct object of testwhen desired tests (for examples, the anticancer agent sensitivity testsas mentioned above, co-culturing tests, and various genetic diagnoses)are carried out, or also using it as a sample to be subjected to aseries of steps comprising the above desired culture and desired test.Among them, particularly the present invention culturing process canfavorably be applied to a step that is a preceding stage of anticanceragent sensitivity tests. The present invention culturing process canfavorably be applied also to all other objects and steps to such anextent that they can be carried out sufficiently.

Next, the culture medium as mentioned in the present invention has aproliferating action and physiological activity-retaining action on theanimal cells, and further has a killing action and/ormultiplication-inhibition action on bacteria.

The aforementioned proliferating action may be at least an action ofmaintaining the cell number of such as the aimed animal cells (cellssampled for biopsy) or animal cell lines without reducing it, favorablyan action of increasing the cell number.

The aforementioned physiological activity-retaining action may be atleast an action of giving some physiological activity to such as theaimed animal cells (cells sampled for biopsy) or animal cell lines stilleven after the culture, favorably an action of retaining or improvingthe same physiological activity as is when sampled from living bodytissue still after the culture. In specific examples thereof, animalcells (cells sampled for biopsy) or animal cell lines, resultant fromculture, have reactivity to such as agents when the cells are used forsuch as tests for sensitive to agents.

The killing action and/or multiplication-inhibition action on bacteriais favorably an action effect which prevents the culture using thepresent invention culturing process or another culture as carried outafter this culture, from becoming uncontinuable because of thecontamination with bacteria. At the same time, a conventional washingstep of washing cells after the cells are sampled from a living body (astep of removing bacteria and various germs) can be simplified by thisaction effect. Therefore, the loss of the cell number can be decreasedwhen the washing is carried out. Accordingly, as a result, even if thecell number of cells as sampled is extremely small, there is no loss ofthe cell number, and the activity can be maintained or proliferated, tosuch an extent that the cells can be used for sensitivity tests, in astate of no unfavorable influence because of the contamination. Inspecific examples thereof, the success rate in the case of carrying outtests such as anticancer agent sensitivity tests can be raised to higherthan before. Incidentally, the bacteria as mentioned herein mainlyincludes bacteria ordinarily living in vivo (for example, Escherichiacoli), which have mingled when cells are sampled from living bodytissue, especially from tissue such as digestive apparatus, and alsoincludes such as bacteria which have mingled from such as air when theculturing procedure is carried out. In addition, in order to make theculture medium have this action effect, 1) such as some agent may beadded to the culture medium, 2) the composition of the culture medium ofthe culture medium itself may be adjusted so that such as multiplicationof the aimed bacteria will be inhibited, thereby obtaining the sameeffect; and 3) the temperature and pH are adjusted so that themultiplication of aerobic bacteria can be inhibited. There is especiallyno limitation. However, among these, it is favorable to make the culturemedium have the aforementioned action effect by adding antibiotic agentsincluding antibiotics as the agents.

The aforementioned antibiotic agents are not especially limited, butspecific examples thereof include at least two members selected from thegroup of antibiotics consisting of cell-wall-synthesis inhibitors,protein-synthesis inhibitors, nucleic-acid-synthesis inhibitors, andantifungal agents. Among these, it is favorable to include at least onemember selected from the group consisting of the cell-wall-synthesisinhibitors. Examples of the cell-wall-synthesis inhibitors favorablyinclude: β-lactam series, such as penicillin series, cephem series,monobactam series, and carbapenem series; phosphomycin; vancomycin; andTeicoplanin. Examples of the protein-synthesis inhibitors include:aminoglycoside series; macroride series; lincomycin series; tetracyclineseries; and chloramphenicol series. Examples of thenucleic-acid-synthesis inhibitors include: ST synthetic compounds, andsynthesized antimicrobial agents, such as pyridocarboxylic acid series,quinolone series, and rifampicin. Examples of the antifungal agentsfavorably include polyene series.

As to the antibiotic agent, only the two antibiotics may be selected andused, or at least three thereof may jointly be used. When at least threeare selected and jointly used, there is no especial limitation withregard to examples of favorable combinations thereof. Specific examplesinclude a combination of four kinds comprising penicillin, kanamycin,fungizone and vancomycin. As to the concentration of each antibiotic asused, the concentration of penicillin is favorably in the range of 125μg/ml to 2 mg/ml, more favorably 250 μg/ml to 2 mg/ml, still morefavorably 500 μg/ml to 2 mg/ml, particularly favorably 1 to 2 mg/ml. Theconcentration of the kanamycin is favorably in the range of 64 μg/ml to2 mg/ml, more favorably 125 μg/ml to 2 mg/ml, still more favorably 250μg/ml to 2 mg/ml, particularly favorably 0.5 to 2 mg/ml. Theconcentration of the fungizone is favorably in the range of 0.32 to 5μg/ml, more favorably 0.64 to 5 μg/ml, still more favorably 1.25 to 5μg/ml, particularly favorably 2 to 5 μg/ml, most favorably 2.5 to 5μg/ml. The concentration of vancomycin is favorably in the range of 0.01to 3 mg/ml, more favorably 0.1 to 2 mg/ml, still more favorably 0.25 to2 mg/ml, particularly favorably 0.5 to 2 mg/ml, more particularlyfavorably 1 to 2 mg/ml, most favorably 2.0 mg/ml. When the culturemedium in the present invention culturing process includes an antibioticagent having the kind (or, its combination) in the range of theconcentration as used in the above way, the culture medium does not haveunfavorable influences such as toxicity to animal cells, and can displaysufficient action effects (killing action and/ormultiplication-inhibition action) on bacteria as targets.

For the same reason, Examples of favorable other combinations using atleast three kinds of the above antibiotic agents are not especiallylimited. Specific examples thereof favorably include: 1) penicillin,gentamycin, (or minomycin), vancomycin, and fumgizone; 2) cephamycin (orcephalosporin), kanamycin (gentamycin or minomycin), vancomycin, andfungizone; 3) penicillin, kanamycin, (or gentamycin, minomycin,cephamycin, or cephalosporin), phosphomycin, vancomycin, and fungizone;4) penicillin, kanamycin (or gentamycin, minomycin, cephamycin, orcephalosporin), ST synthetic compounds (or othernucleic-acid-synthesizing agents), vancomycin, and fungizone. As to theconcentration of the gentamycin, minomycin, cephamycin, andcephalosporin as used in the above other favorable combination example,the concentrations of the gentamycin, minomycin, cephamycin, andcephalosporin are favorably in the ranges of: 0.5 to 1.0 mg/ml, 0.1 to0.2 mg/ml, 1 to 2 mg/ml, and 1 to 2 mg/ml respectively.

Favorable examples of the usable culture medium in the present inventionculturing process include serum culture mediums containing serum, andserum-free culture mediums being free from serum, and either can be usedin the present invention. Favorable examples of the serum include FBS(fetal bovine serum), FCS (fetal calf serum), HS (horse serum), orunworkable FBS, and the FBS is more favorable.

The serum-free culture mediums are characterized by containing no serum,and on the other hand conventional culture mediums used for cell culturecontain serum as a component. The serum-free culture medium is preparedby combining various chemical substances necessary for culturing, otherthan serum. Particularly, the serum-free culture medium can suppress theproliferation of fibroblast cells effectively and can inhibit thecontraction of collagen gel. In the case where the serum-free culturemedium, the specific components and their proportions thereof may bedetermined as needed, but the composition having good proliferativity ofthe aimed animal cells (for example,. cancer cells) and inhibiting theproliferation of other cells is favorable.

On the other hand, as to the serum culture medium, usually that whichhas a serum concentration of about 0.001 to about 5.0 % is favorablyused, but good results are sometimes obtained even by using that ofwhich the serum concentration is in the range of about 5 to about 20%.

In addition, even if the usable culture medium in the present inventionculturing process is made to contain dextran sulfate, the contraction ofcollagen gel can be inhibited. The reason therefor is because thedextran sulfate also has the effect of inhibiting the proliferation offibroblast cells in the same way as of the serum-free culture medium,and the dextran sulfate can be used instead of preparing the serum-freeculture medium. It is favorable that the ratio of the dextran sulfate asadded to the culture medium is in the range of 1.5 to 100 μg per 1 ml ofthe culture medium, and it is more favorable that the ratio of thedextran sulfate having a molecular weight of not less than 50,000 is inthe range of 3.0 to 100 μg, and it is still more favorable that theratio of the dextran sulfate having a molecular weight of not less than500,000 is in the range of 1.5 to 50 μg. Whichever of theabove-mentioned serum culture medium and serum-free culture medium maybe used, the dextran sulfate can be added thereto.

The sample (culture sample) as used in the present invention culturingprocess, is a sample containing animal cells sampled from living bodytissue. However, these animal cells, that is, primary cells, arefavorably primary cells (what are called low invasive sampling cells)obtained by a low invasive sampling method. In addition, these lowinvasive sampling cells are particularly favorably cells that areexcision or test-puncture sampled for the purpose of such as medicaldiagnosis, curing of diseases, and judgment of prognosis, and isobtained from such as various biopsy cells, materials below thoracoscopeor laparoscope, ascites, and malignant pleural fluid.

After the sampling, the sample (culture sample) as used for the cultureemploying the present invention culturing process is favorably subjectedto dispersing treatment, or separating and dispersing treatmentsbeforehand when the occasion demands.

The separating treatment is not especially limited, but it is favorablythe treatment such that a culture sample is divided into small pieces inorder to enhance the effect of the dispersing treatment that is carriedout after the separating treatment. More particularly, this treatment isfavorably mechanically separating treatment. Among these, thefine-cutting treatment with such as cesisers, tweezers, and razors isfavorable.

The dispersing treatment is not especially limited, but favorableexamples thereof include treatment of removing cells other than theaimed animal cells in living body tissue (for example, cancer cells) orintercellular substances, or treatment of removing other substances thatcan inhibit subsequent various tests. More particularly, thesetreatments are favorably enzymatic dispersing treatments, and such ascollagenase, hyaluronidase, DNase, elastase, and dispase are favorableas the enzyme as used then.

The culture sample as used for the above-mentioned dispersing treatmentand/or separating treatment may be a slight amount of 0.001 to 1 g oftissue. When this dispersing treatment and/or separating treatment arecarried out, the animal cells (primary cells) are isolated from livingbody tissue.

Such as treatment conditions of the aforementioned dispersing treatmentand/or separating treatment are provided in the following ways 1) to 3),depending upon the size of the aforementioned tissue lump as sampledfrom living body. 1) In the case where the above tissue lump has a sizeof not smaller than 3 mm cube, it is favorable to carry out themechanical separation and enzymatic dispersion. 2) In the case where theabove tissue lump has a size of 0.5 to 3 mm (but not including 3 mm)cube and smaller than 3 mm cube, the mechanical separation is notneeded, but it is favorable to carry out only the enzymatic dispersion.3) In the case where the above tissue lump has a size of smaller than0.5 mm cube and is as small as a solid tissue portion that is notconfirmed with the naked eye, neither the mechanical separation norenzymatic dispersion may be needed. The further detailed treatmentconditions of the above 1) to 3) are described in the below-mentionedexamples of some preferred embodiments.

In the culturing process according to the present invention, the cultureis favorably carried out on a surface of a supporting base, and theaforementioned supporting base favorably has a layer, including anextracellular matrix, as a cell adhesion factor. In the presentinvention, conventional culture methods (e.g. a monolayer culturemethod, a coated dish culture method, and a on-collagen-gel culturemethod) can favorably be employed. Among them, a two-dimensionalmonolayer culture method is favorably employed when the culture iscarried out on the surface of the supporting base in the above way (whenthe culture is carried out in a state where the aimed cells are stuck onthe surface of the supporting base). Specifically, a culture sample anda culture medium are placed in a culture container having the supportingbase, and kept under definite environmental conditions, whereby onlyspecific live cells containing the aimed animal cells (e.g. cancercells) are proliferated under conditions where they are stuck on thesurface of the supporting base of the culture container. At this time,such as apparatuses as used and treatment conditions are those as insuch as conventional monolayer culture method.

It is favorable that the surface of the aforementioned supporting basecomprises a material which allows the cells to favorably adhere andproliferate, or is coated with a single layer of a chemical substance ora cell adhesion factor, which allows the cells to favorably adhere andproliferate. Examples of this cell adhesion factor favorably includeextracellular matrixes, such as various types of collagen, fibronectin,laminin, vitronectin, cadherin, gelatin, peptides, and integrin. Thesemay be used either alone respectively or in combinations with eachother. In view of further improving the cell adhesion and cellextension, various collagens are favorably used. Among the variouscollagens, a type-I or type-IV collagen is particularly favorably used.

The shape of the culture container having the aforementioned supportingbase is not especially limited, and it may be any shape. However, forexample, a dish and a tube are favorable. The dish is not especiallylimited, but examples thereof favorably include: dishes for culturingcells, such as plastic dishes and glass-made Petri dishes; multiplates,such as micro wellplates or micro titerplates having such as 6, 12, 24,48, or 96 wells; flasks for culturing cells; and culture plates, such ascover slips and cell disks. The tube is not especially limited, butexamples thereof favorably include flat-bottomed or round-bottomed tubecontainers. Examples thereof more favorably include flat-bottomed tubecontainers obtained by cutting a portion of a tube as shown in FIGS. 13and 14,. so that the containers will have a moderately gentle angle tothe central axis of the containers. Incidentally, the cutting face ofthe tube as shown in FIGS. 13 and 14 is a flat around-wall. Among theculture containers having the above exemplified shapes, the tube,especially the tube container having a shape as shown in FIGS. 13 and 14is especially favorable at the following points. It has a surface shapeand. surface area of the around-wall which is fitted for culturingbiopsy cells (In the present invention, when the “surface area” or“bottom area” is mentioned as to the supporting base, the area isdefined as an area of a portion of sticking and culturing cells on thesupporting base.), and besides has an excellent coatability of the celladhesion factor. In addition, the sudden pH change of the culture mediumcan be suppressed. Therefore, the initial adhesion of the aimed cancercells and the maintenance of the cell activity are excellent, and thetube can stabilize activities of antibiotic agents. Furthermore, livingadhered cells can be collected by such as subjecting them exactly tocentrifugal treatment, and thereby the high collection ratio can beachieved and the loss can be greatly decreased when the collection iscarried out. Accordingly, for example, there are particularly advantagesin that the culture having a high density can also be carried out in avery stable culture state.

The surface area of the aforementioned supporting base (area of aportion where cells are stuck and cultured) is favorably in the range of0.01 to 25.0 cm², more favorably 0.5 to 10.0 cm², still more favorably2.0 to 6.0 cm². In addition, as to the tube container as shown in FIGS.13 and 14, the area of the aforementioned cutting face to be a flat faceis particularly favorably 5.5 cm², and various excellent effects whenthis tube container is used can remarkably be displayed. In the casewhere the surface area of the aforementioned supporting base is smallerthan 0.01 cm², it is difficult to seed cells onto the supporting baseand it is limited to proliferate cells. In addition, in the case wherethe surface area of the aforementioned supporting base is larger than25.0 cm², the cell density is lowered when the cells-seeding is carriedout, and the interaction between the cells does not act sufficiently.Therefore, the initial adhesion is lowered.

As is mentioned in the above way, when the culturing process accordingto the present invention is a culturing process carried out on thesurface of the supporting base having the layer which includes theextracellular matrix as the cell adhesion factor, the unnecessarycomponent which does not adhere to the surface of the supporting basecan be removed in the culture sample, and only the live cells adheringto the surface of the supporting base can favorably be collected byremoving the culture medium in the culture container after the culture.Particularly, such as tumor-dead cells, their lumps, lumps of fibroblastcells, undigested materials of enzymes, blood cells, and lymphocytes canfavorably be removed. In the collection of live cells adhering to thesurface of the supporting base, the means such as EDTA-trypsin treatmentcan be applied and is favorably carried out after the above monolayerculture method.

In addition, in the culturing process according to the presentinvention, a collagen gel layer can be used when the aforementionedculture is carried out on the gel. The material of the collagen gellayer may be the same collagen material to form the below-mentionedglobular collagen gel. The thickness of the collagen gel layer may besuch a thickness as not to directly stick the aimed animal cells (e.g.cancer cells) to the surface of the supporting base. The aimed animalcells (e.g. cancer cells) as adhered to the surface of the collagen gellayer can easily be separated from the surface of the supporting base bycollagenase treatment, and then collected.

In this way, if the collagen gel layer is beforehand formed on thesurface of the supporting base, and the culture sample including theaimed animal cells (e.g. cancer cells) is cultured, and only the livecells adhering to the surface of the collagen gel layer is collected bythe collagenase treatment, the proliferation of the aimed animal cells(e.g. cancer cells) on the collagen gel is more favorably carried outthan the culture in a state of directly sticking the animal cells (e.g.cancer cells) on the surface of the supporting base. The collagenasetreatment damages little live cell when proliferated live cells arecollected. This is because the collagen gel layer itself whereon thelive cells stick are enzymatically decomposed prior to the action to thelive cells in the collagenase treatment, and thereby they have littleunfavorable influence upon the live cells.

The process for culturing an animal cell, according to the presentinvention, may be a culturing process including only the above-mentionedculture step, or a culturing process further including other varioussteps.

Although the culturing process further including other various steps isnot especially limited, for example, favorable is a culturing processcomprising: a preliminary culture step using the above-mentionedculturing process; a cells-seeding step subsequent to this preliminaryculture step; an embedding step; and a proliferative culture step.

The aforementioned cells-seeding step is a step of dispersing the animalcells, which are contained in culture samples collected in thepreliminary culture step, into a collagen solution.

The aforementioned embedding step is a step of placing the a droplet ordroplets of the collagen solution, resultant from the cells-seedingstep, on a surface of a supporting base material and allowing thedroplet or droplets to gel to form and fix on the surface of thesupporting base material a globular collagen gel having a convexsurface.

The aforementioned proliferative culture step is a step of contactingthe collagen gel, resultant from the embedding step, with a liquid as aculture medium and culturing the animal cells.

The culturing process may further comprise an evaluation step after theproliferative culture step.

As is mentioned in the above way, if the culture sample including theaimed animal cells (e.g. cancer cells) is beforehand cultured on asurface of other supporting base in the preliminary culture step, andeven if the cell number (absolute amount) of the aimed animal cells(e.g. cancer cells) as obtained by sampling from a living body is small,the cell number can sufficiently be increased and maintained due to theabove-mentioned effects. Accordingly, even if the number of the test asdesired to carry out after the culture is large and the division of thecell number is necessary, the sufficient number of the aimed animalcells (e.g. cancer cells) can be embedded in the globular collagen gelfor each test. Therefore, a more correct test having a higher successrate can be carried out.

Hereinafter, the aforementioned cells-seeding step, embedding step,proliferative culture step, and evaluation step are specificallyexplained.

[Cells-seeding Step]:

A collagen solution used for forming the globular collagen gel(s) maycontain a common collagen material conventionally used in various animalcell-embedded culture methods, a high-molecular material such aspolysaccharides and other extracellular matrixes, and a liquid material.For example, the collagen used is favorably acid-soluble type-Icollagen. Various components other than collagen, which are necessaryfor culture, may also be added to the collagen solution. The collagensolution preferably has the composition of a buffer solution to matchwith or approximate to physiological conditions of the aimed animalcells (e.g. cancer cells). As to the cancer cells, the solution isfavorably buffered to a pH of 6.2 to 7.6, more favorably 6.8 to 7.4,while the ionic strength is favorably set within the range of 100 to 180mmols, more favorably 140 to 160 mmols. Conventional methods may beapplied for mixing the culture sample into the collagen solution. Thedensity of the aimed animal cells (e.g. cancer cells) to seed into thecollagen solution is favorably in the range of about 10² to about 10⁷cells/ml. Especially for the primary culture such as the culturingprocess according to the present invention, the density is favorably inthe range of about 1 to about 10⁶ cells/ml.

The collagen concentration and the viscosity of the collagen solutioninfluence the structure of the later-mentioned globular collagen gel.The specific quantitative conditions vary dependently on conditions suchas the purpose of the test, but the collagen concentration is favorablyin the range of 0.1 to 2.0 wt. %. In the case where the concentration istoo high, the viscosity as mentioned below is high. On the other hand,in the case where the concentration is too low, the globular shape isdifficult to maintain. The viscosity is favorably in the range of 50 to2,000 centipoise, more favorably 100 to 1,000 centipoise. In the casewhere the viscosity is too low, the cells precipitate in the collagensolution, come into contact with a surface of the supporting basematerial, and proliferate in the form of a monolayer, thus making itimpossible to make an accurate evaluation of various effects in testsafter the culture, such as effects of anticancer agents. On the otherhand, in the case where the viscosity is too high, the collagen solutionis difficult to handle. In addition, the gel strength upon gelation ofthe collagen solution also affects the performance, and therefore thecollagen as used preferably has a gel strength in the range of about 50to about 1,000 g, more favorably in the range about 50 to about 700 g,and still more favorably in the range. 100 to 500 g. The value of thegel strength is that measured according to JIS. In the case where thegel strength is too low, the globular collagen gel peels off from asurface of the supporting base material during tests, or the gel tendsto contract. On the other hand, the gel strength is too high, theproliferation of the, aimed animal cells (e.g. cancer cells) isinhibited.

[Embedding Step]:

The aforementioned supporting base material is not limited with itsmaterial and structure so long as it has a surface capable of fixing acollagen gel thereto. Examples of the usable supporting base materialinclude culture dishes such as Petri dishes and multidishes, flasks, andother conventional culture containers. In addition, examples of theusable supporting base material further include culture plates such asglass or plastic cover slips and cell disks. The surface of thesupporting base material is usually smooth and flat, but lines includingframe-forming ridges and grooves to control the spread of the placeddroplet of the collagen solution may also be formed on the surface ofthe supporting base material. It is favorable that these culturecontainers and supporting base materials are optically transparent.

If a droplet or droplets of the collagen solution containing a dispersedculture sample is placed on a surface of the supporting base material,such as the surface tension of the collagen solution acts to form awater-like droplet or droplets on the surface of the supporting basematerial. As a result, a globular collagen gel or gels with a sphericalconvex surface can be formed on the surface of the supporting basematerial.

The shape of the globular collagen gel differs depending on factors suchas the viscosity and temperature of the droplet of the collagen solutionwhich is placed, the amount of each droplet of the collagen solutionwhich is placed, the wettability of the surface of the supporting basematerial, and other, conditions. In addition, it is preferable to setthe gel to the desired shape depending on the test method to be employedincluding, for example, photography conditions when photographing theglobular collagen gel for the image analysis.

As to modes of placing the globular collagen gel on the supporting basematerial, one or more droplets of one collagen gel may be placed, oreach droplet of at least two collagen gels may be placed. In addition,when the at least two collagen gels are cultured simultaneously, thegels may be in contact with each other on their partial or entireinterface.

If the globular collagen gel having a convex surface is used, theglobular collagen gel has a much smaller volume than a layered collagengel, and thus, even when only a small amount of cancer cells is obtainedfrom living body tissue, the cells can be seeded to a suitable andsufficient density. In addition, a wide area of the convex surface comesinto contact with the culture medium, thus allowing for very efficientuptake of nutrients by the cells and excretion of waste from the cells.Furthermore, if the size of the culture container is selected, theamount of the culture medium coming into contact with the collagen gelcan relatively be increased. The culture method of the present inventionfar more greatly improves the cell proliferativity than conventionalmethods in which a collagen gel layer is formed over the entire internalbottom of a culture dish and nutrient and waste are exchanged only at aflat surface of the collagen gel layer.

In addition, the globular collagen gel is fully fixed onto a surface ofa supporting base material, because the globular collagen gel is formedby placing a droplet or droplets of a collagen solution containing theaimed animal cells (e.g. cancer cells) onto the surface of a supportingbase material such as a culture dish, and then gelating the droplet(s).Consequently, results of culturing the aimed animal cells (e.g. cancercells) may be observed or photographed while more easily specifyingpositions of the cells after culturing than results obtained byculturing in a layered collagen gel. In addition, the method for placinga droplet or droplets of the collagen solution, for example, may includedropping the collagen solution from above the surface of the supportingbase material, or gently placing the collagen solution into the form ofa globular water-like droplet or droplets using a pipette near thesurface of the supporting base material.

Specifically, under normal test conditions, the size of each globularcollagen gel is made to be in the range of 3 to 300 μl, favorably 3 to150 μl, in terms of the volume of a droplet of the placed collagensolution. For practical use, it is favorably in the range of 5 to 100μl, more favorably about 15 to about 50 μl. As is in the culturingprocess according to the present invention, if primary cells arecultured, the gel is favorably about 30 μl. In addition, the height ofthe globular collagen gel is favorably adjusted to about not more than 2mm. The number of the formed collagen gel globules and their placingintervals on the surface of the supporting base material may optionallybe varied as desired to match the measurements of the globular collagengel and the structure of the supporting base material.

Collagen gels as used for microscopic observation and image analysishave needed to have a high transparency hitherto. However, in theprocess according to the present invention, collagen gels even with arelatively low transparency can be used. Specifically, collagen gelswith a transmittance of 1 to 95% for 400 nm light can be used.

A test even using collagen gels with a low transmittance within theabove-mentioned range can satisfactorily be carried out without beingaffected by turbidity if the collagen gels are subjected to thebelow-mentioned stain treatment.

Samples as mixed into the globular collagen gel may be various cells,such as cancer cells and normal cells, and besides may be compoundswhich act on the cells, for example, calcium, inorganic salts such ascalcium phosphate, lipids, carbohydrates, and proteins.

[Proliferation Culture Step]:

The formed globular collagen gel needs to be kept in a gel state atleast until the culture finishes. Even a three-dimensional globularcollagen gel becomes flat and dry if it loses moisture due to drying.Once it is dried, it is difficult to restore to its originalthree-dimensional shape even if it is brought into contact with water.Thus, the globular collagen gel is preserved, after its formation untilsupplying such as the culture, so that it may not excessively dry. Solong as the globular collagen gel is in contact with the culture medium,there is no possibility that the gel may be dried. After the completionof the culture, the fixing and drying of the globular collagen gelpresents no problem for evaluating culture results.

If a culture container having the supporting base material is such as aculture dish, the culture medium may be brought into contact with asurface of the supporting base material by simply pouring the culturemedium into the container to cover the surface of the supporting basematerial. If a culture container having the supporting base material isa culture plate, the culture medium may be poured in under conditionswhere the culture plate is placed in another culture container.

Although the culture medium usable in the proliferative culture step isnot especially limited, it may fitly be selected from various knownculture mediums depending upon test conditions. However, the culturemedium is favorably has such a composition as is suitable forproliferating the aimed animal cells (e.g. cancer cells) and cansuppress the proliferation of other cells. In the present invention, forexample, it is favorable that the culture medium is a culture medium asused in the preliminary culture step.

The amount of the culture medium as used in the proliferative culturestep is enough to cover the globular collagen gel for a satisfactorydegree of culturing. In the case where the number of the aimed animalcells (e.g. cancer cells) as embedded in the globular collagen gel islarge, the amount of the culture medium needs to be increased or theintervals of exchanging the culture medium during the culture needs tobe shortened.

In addition, in the case where interactions of cells are observed insuch as co-culture tests, a collagen layer which contains tested cellsinstead of the culture medium can be placed in the layered form over theentire container, or a globular collagen in which other cells aredispersed can be placed in the same culture container.

The culture in the proliferation culture step is favorably carried outby keeping the culture medium in contact with the globular collagen gelas fixed to the surface of the supporting base material, for a definiteperiod of time under environment conditions such as in a thermostatincubator or a CO₂ incubator. In order to such as test the sensitivityto an anticancer agent after the culture, it may be brought into contactwith the globular collagen gel as fixed to the surface of the supportingbase material, at an appropriate stage before or during the culture.Examples of the favorable method thereof include a method in which theanticancer agent is added to the culture medium, or a method in whichthe culture medium is exchanged with a culture medium to which theanticancer agent is added. Procedures and conditions for bringing theanticancer agent into contact with the collagen gel may be optional. Inaddition, when effects of agents other than the anticancer agent aretested, such agents may be brought into contact with the collagen gel inthe same manner as the anticancer agent. When effects of a change oftemperature, for example, by heating, or effects of radiations or thelike are tested, the globular collagen gel may be exposed under thoseenvironment conditions for a definite period of time.

[Evaluation Step]:

Depending on culture conditions, the aimed animal cells (e.g. cancercells) in the globular collagen gel are proliferated or dead after thecompletion of the culture. Means of evaluation as in conventionalculture methods may be applied in order to evaluate culture results,such as the proliferation state of the aimed animal cells (e.g. cancercells). For example, the number of the formed colonies or cells may becounted by visual observation using a microscope, or an image obtainedby photographing or imaging may be analyzed. Various characteristics ofthe aimed animal cells (e.g. cancer cells) existing there may beobserved and evaluated while the globular collagen gel is fixed to thesurface of the supporting base material.

When the image analysis is carried out as a method for evaluatingculture results, such as the placement form of the globular collagen gelon the surface of the supporting base material or the structure of theculture container is set so as to facilitate the photographing and imageanalysis. A supporting base material with the aforementioned linesincluding ridges and grooves to restrict the shape is favorably used inorder to precisely set the shape and position of the globular collagengel on a surface of the supporting base material. The images of theaimed animal cells (e.g. cancer cells) and their colonies can bedistinguished from other images such as of fibroblast cells by utilizingimage analysis in which photographed images are electronically processedand analyzed using such as a computer, thus the proliferation state ofthe aimed animal cells (e.g. cancer cells) can accurately be evaluated,because cells as cultured under embedded conditions in the globularcollagen gel show specific proliferation morphology.

After culturing using the globular collagen gel, if only live cells inthe globular collagen gel are selectively stained and if its results areevaluated, then live cells can accurately be distinguished from deadones in the aimed animal cells (e.g. cancer cells), thus allowing anaccurate evaluation of the proliferation state of the aimed animal cells(e.g. cancer cells) being studied in the culture test. The stain methodmay be a conventional method for staining various cells, so long as itis a method capable of selectively staining live cells as distinct fromother substances including dead cells. Particular staining agents andstaining conditions may be according to conventional methods. Forexample, NR stain methods or latex particle stain methods in whichphagocytic activity of cells is utilized, SDI methods or FDA stainmethods in which enzyme activity in cells is utilized, or other stainmethods using a fluorescent reagent, or the like may be used.

An example of the treatment for selectively staining only live cells isan NR stain treatment, which uses NR (neutral red) stain as the stainingagent. Stain conditions may be the same as in conventional NR stain. Inthis NR treatment, if a pigment is fixed into cells and if the globularcollagen gel is then dried, the dried globular collagen gel reflectingculture results by the stained state can be preserved for a long time,and the procedure of evaluation such as by image analysis provide thesame accurate analysis whenever the evaluation procedure may be made. NRstain is a preferable method for selectively staining only live ones ofthe aimed animal cells (e.g. cancer cells), but if left to stand afterstaining, an NR-staining agent as incorporated into live cells eluteswithin a short period, thus making an evaluation impossible except justafter staining. If then, as mentioned above, the pigment is fixed intocells by such as formalin fixation, it becomes possible to temporarilyprevent elution of the staining agent. If the globular collagen gel isthen washed with water and dried, there is no possibility ofdegeneration or degradation. Means for drying, for example, may bewater-absorption using filter paper, air-drying, or forcible drying byheating to a range of about 10 to about 50 ° C. The dried collagen gelprepared by removing the moisture from the globular collagen gel has aflat, sheet-like structure. The dried globular collagen gel cansuccessfully be sealed using sealants such as Crystal/Maunt (produced byBiomeda).

In addition, in the case where image analysis is performed, thephotography procedure is much easier by setting the focus on the aimedanimal cells (e.g. cancer cells) in a flat, dry collagen gel rather thanon the aimed animal cells (e.g. cancer cells) in a three-dimensional,globular collagen gel, allowing a more focused image and thus a moreaccurate evaluation of culture results.

FDA stain treatment is another treatment for selectively staining onlylive cells. This treatment is a method including measuring fluorescentcoloring as produced by a reaction of an FDA (Fluorescein Di Acetate)staining agent upon live cells followed by exposure to excitatory light.Basic apparatuses and conditions for tests may be the same as inconventional FDA stain methods, but in the present invention theirradiation amount of excitatory light and the measurement environmenttemperature are set to within the ranges given later. The in-vivo-likemorphology of colonies that fluorescently colored cells form in theglobular collagen gel can be quantified and evaluated by image analysis.The FDA stain method is a method which allows satisfactory selectivestain of only live ones of the aimed animal cells (e.g. cancer cells).However, since conventional FDA stain methods include irradiatingrelatively strong excitatory light or carrying out the measurement atabout normal temperature, these methods have disadvantages, for example,in that the activity of the aimed animal cells (e.g. cancer cells) islowered. However, such disadvantages may be reduced if the irradiationamount and the temperature conditions are kept within the ranges givenbelow. As a result, prior to culturing the aimed animal cells (e.g.cancer cells) or at any stage during the culture, the evaluation of theculture state by an FDA stain method, and thereafter the continuation ofthe culture can be carried out. In other words, the continuousevaluation of the culture state can be carried out. In particular, ifthe culture method under embedded conditions in the globular collagengel is combined with the evaluation method utilizing image analysis, ahighly precise measurement and evaluation can be made even underconditions where the irradiation amount of excitatory light is small andwhere the fluorescent coloring is weak, thus such a combined method ispreferred.

The conditions for measurement after the FDA stain are as follows: theirradiation amount of excitatory light is in the range of 1×10⁰ to 1×10⁷lux sec, favorably 1×10¹ to 1×10⁵ lux-sec, and the measurementenvironment temperature in the range of 1 to 15° C., favorably 8 to 12°C.

If the colorimetric analysis as another method for evaluating cultureresults is carried out using a coloring reagent which selectively causesa color reaction due to metabolic activity of live cells as embedded inthe globular collagen gel, then culture results can comparatively easilybe evaluated even without using complicated equipments such as imageanalyzers. The accuracy of the evaluation is sufficient in practicalapplication, particularly if a culture sample with little contaminationby fibroblast cells, or an established cell line, is used as a culturesample or if the contamination or proliferation of the fibroblast cellsis suppressed in any of the various manners as mentioned above. Inaddition, if the coloring agent is soluble in water, the evaluation cancontinuously be carried out, because the water-soluble coloring agentgives no unfavorable affection to the animal cells (e.g. cancer cells)as same as the FDA stain.

Specific methods and conditions for the step including theaforementioned calorimetric analysis may be the same as in culture testsfor other cells. Examples of the coloring reagent as used include an ABcoloring reagent (trade name: Alamar Blue, produced by Alamar BioscienceCo., Ltd.), a WST-1 coloring reagent, an XTT coloring reagent and a MTTpigment reductant.

As a method in which the measurement can be carried out even if cellsare dead, there is a method including selectively staining substances ascontained in cells in the globular collagen gel, thus evaluatingresults. The stain methods as employed may be a variety of conventionalbiochemical stain methods so long as they allow the selective stain ofsubstances as contained in cells. Specific staining agents andconditions may be conventional ones, for example, staining reagents asused for staining tissue can be used.

Specific examples of the staining agents include hematoxylin, Giemsa'ssolution, pigments such as Crystal Violet, ethidium bromide which stainsnucleic acid, reagents which stain components such as proteins,carbohydrates and lipids, reagents which stain substances in cellmembranes, reagents which stain specific parts such as cytoskeletons,antibodies against specific antigens, DNA probe.

If substances as contained in cells in the globular collagen gel areselectively stained and if its results are evaluated, the productivityof specific substances from cells can clearly be distinguished whetherthe cells are alive or not, thus the proliferation state of the animalcells (e.g. cancer cells) which are objects of culture tests canaccurately be evaluated from the substance productivity of the cells.

In addition, live cells or cell components can be stained doubly ortriply. Various actions of cells can be evaluated for an identicalsample by multiple staining of the cells.

Furthermore, the globular collagen gel may be separated from the surfaceof the supporting base material, for example, by dissolving the gel,thus evaluating culture results.

An example of the methods is a method including: quantitativelyanalyzing substances as contained in cells in the globular collagen gel;and evaluating its results. The quantitative-analysis methods asemployed may be a variety of conventional biochemical ones so long asthey allow quantitative analysis of substances as contained in cells.Specific methods for quantitative analysis may be in accordance withconventional ones, for example, a commassie brilliant blue color methodor Lowry method for quantitatively analyzing proteins, a DABAfluorescent coloring method for quantitatively analyzing nucleic acid, aluciferin luminescence method for measuring ATP, or various methods forquantitatively analyzing carbohydrates.

If substances as contained in cells in the globular collagen gel arequantitatively analyzed and if its results are evaluated, theproductivity of specific substances from cells can clearly bedistinguished whether the cells are alive or not, thus the proliferationstate of the animal cells (e.g. cancer cells) which are objects ofculture tests can accurately be evaluated from the substanceproductivity of the cells.

In addition, another method includes quantitatively analyzing substancesas secreted from cells in the globular collagen gel to a culture medium,and evaluating its results. The quantitative analysis methods asemployed may be a variety of conventional biochemical ones so long asthey allow quantitative analysis of substances as secreted from cells.Specific methods for quantitative analysis may be in accordance withconventional ones, for example, various methods such as methods forquantitatively analyzing lactic acid and the like which are typicalmetabolic products (waste).

If substances as contained in cells in the globular collagen gel arequantitatively analyzed and if its results are evaluated, theproductivity of specific substances from cells can clearly bedistinguished whether the cells are alive or not, thus the proliferationstate of the animal cells (e.g. cancer cells) which are objects ofculture-tests can accurately be evaluated from the substanceproductivity of the cells.

The kit for culturing animal cells, according to the present invention,comprises the following constituent articles: a collagen solution; aconcentrated culture medium; a reconstituting buffer solution; an enzymefor dispersing cells; a tube for a culture supporting base; a culturemedium for preliminary culture a serum-free culture medium; and acell-dyeing agent.

The kit for culturing animal cells, according to the present invention,can favorably be used for carrying out the aforementioned process forculturing animal cells according to the present invention.

Hereinafter, the aforementioned respective constituent articles areexplained with citing specific examples.

The collagen solution is not especially limited, but specific examplesthereof favorably include acid-soluble type-I or type-IV collagens, orpepsin-soluble type-I or type-III collagens. Among them, theacid-soluble type-I collagens are more favorable.

The concentrated culture medium is not especially limited, but specificexamples thereof favorably include culture fundamental medium formammalian cells, such as McCoy's SA, RPMI-1640, D-MEM, MEM, MCDB-131,Ham's F-12, D-MEM/F-12, and Medium-199. Among them, the Ham's F-12,D-MEM, and D-MEM/F-12 are favorable.

The reconstituting buffer solution is not especially limited, butspecific examples thereof favorably include phosphoric-acid buffersolutions of which the pHs are adjusted to around pH 7.4, more favorablya 50 mM-NaOH solution containing 260 mM-sodium bicarbonate and 200nM-HEPES, as used in Example 1 below.

The enzyme for dispersing cells is not especially limited, but specificexamples thereof favorably include collagenase, pronase, trypsin,dispase, elastase, hyaluronidase, mucinase, and DNase. Among them, morefavorable is a complexed enzyme including such as the collagenase,dispase, elastase, hyaluronidase, and DNase.

The tube for a culture supporting base is favorably the above-mentionedtube container as shown in FIGS. 13 and 14.

The culture medium for preliminary culture is not especially limited,but specific examples thereof favorably include culture mediums obtainedby adding fetal bovine serum (FBS) to Ham's F-12, D-MEM, or a D-NEM/F-12mixed solution so that the concentration of the fetal bovine serum willbe in the range of 5 to 20%, and culture mediums obtained by furtheradding various growth proliferation factors to this culture, morefavorably culture mediums containing such various antibiotics asmentioned in Example 1 below, in these culture mediums.

The serum-free culture medium is not especially limited, but specificexamples thereof favorably include culture mediums obtained by addingvarious growth proliferation factors to Ham's F-12, D-MEM, or aD-MEM/F-12 mixed solution, more favorably culture mediums containingsuch various antibiotics as mentioned in Example I below, in theseculture mediums.

The cell-dyeing agent is not especially limited, but specific examplesthereof favorably include Neutral Red, Trypan Blue, Alamar Blue, Giemsa,Crystal Violet, FDA, and WT reagents. Among them, more favorable is theNeutral Red which utilizes phagocytosis to lysosome and has highcorrelation to cell life.

The kit for culturing animal cells, according to the present invention,may comprise other constituent articles other than the aforementionedconstituent articles, and they are not especially limited. Specificexamples thereof favorably include medical or curative medicines, suchas washing liquids or preserving liquids for biological tissue (e.g.culture sample as mentioned in the present invention), anticanceragents, anticancer-action promoters, inhibitors for angiogenesis,inhibitors for cancer metastasis, and anti-hormone agents.

Effects and Advantages of the Invention

The present invention can provide a novel process for culturing animalcells, in which, even if the number of cells as sampled for biopsy isextremely small, the proliferation can sufficiently be maintained so asto enable to carry out various tests, and the contamination withbacteria can be inhibited without damaging physiological activity ofcells.

Detailed Description of Preferred Embodiments

Hereinafter, the present invention is more specifically illustrated bythe following examples of some preferred embodiments. However, thepresent invention is not limited thereto.

EXAMPLE 1

<<Anticancer Agent Sensitivity Test>>

Various cancer cells (various cancer cells as oriented from differentorgans), which were cells as sampled for biopsy, were subjected to ananticancer agent sensitivity test by the collagen gel-embedded culturemethod following the procedure as shown in the flow sheet of Table 1.Used as the cancer cells subjected to the test were breast cancer cells,lung cancer cells, colon cancer cells, and pancreatic cancer cells.

Regarding the various cancer cells, there were determined the successnumber and success rate of the primary culture to the number of theperformed tests, and further, only those of which the primary culturehad succeeded were taken as the subjects to carry out the anticanceragent sensitivity test to determine the success number and success rateof the anticancer agent sensitivity test to the success number of theprimary culture. Their results are shown in Table 2. The primary culturemeans the preliminary culture step as also shown in the flow sheet ofTable 1.

In addition, shown in Table 3 from the viewpoint of the success orfailure of the primary culture are, as to the success cases of theprimary culture, as follows: they are the numbers of success cases(cases where evaluation was possible) and failure cases (cases whereevaluation was impossible due to insufficient Cell proliferation) of thesubsequent anticancer agent sensitivity test; and further as to thefailure cases of the primary culture, as follows: the numbers of caseswhere it was impossible for cells to proliferate and cases wherecontamination was caused by such as unwanted bacteria.

Incidentally, as to the culture medium as used in each step in Example1, there is used a culture medium as prepared by adding variousantibiotic agents (penicillin, kanamycin, fungizone, vancomycin) to thefollowing basal medium.

Basal Medium:

DF culture medium (DF: a mixed culture medium containing one volume ofDulbecco modified Eagle (DME) culture medium and one volume of Ham's F12, culture medium)+10% FBS (FBS: fetal bovine serum)+5 μg/ml insulin(product of Sigma Co.)+10 ng/ml EGF (product of Collaborative Co.; EGF(epidermal growth factor))+20 ng/ml hydrocortisone (product of SigmaCo.)

Penicillin Kanamycin:

Two 1 g-vials of penicillin (product of Toyama Chemical Co., Ltd.; forpenicillin injection) and 1 g potency of kanamycin (product of GIBCO BRLCo., Ltd.; sulfuric acid kanamycin reagent) are dissolved into 20 ml ofPBS, and the resultant solution is added to the basal medium in such anamount as gives a concentration of 1% (final concentration ofpenicillin=1 mg/ml; final concentration of kanamycin=0.5 mg/ml) on thebasis of the basal medium.

Fungizone:

Amphotericin B (product of Dainippon Pharmaceutical Co., Ltd.) of 250μg/ml is added to the basal medium in such an amount as gives aconcentration of 1% (final concentration=2.5 μg/ml) on the basis of thebasal medium.

Vancomycin:

An amount of 1 g of vancomycin hydrochloride (product of Wako PureChemical Industries, Ltd.) is dissolved into 10 ml of FBS, and theresultant solution is added to the basal medium in such an amount asgives a concentration of 2% (final concentration=2 mg/ml) on the basisof the basal medium. TABLE 1 <<Anticancer agent sensitivity test flowsheet>> (1) Sampling of test materials (biopsy tumor tissue) ↓ (2)Separating and dispersing treatments of cells ↓ (3) Preliminary culturestep (proliferation and collection of live cells) ↓ (4) Cells-seedingstep for collected cells (preparation of collagen solution) ↓ (5) Stepof embedding a globular collagen gel ↓ (6) Proliferative culture step(proliferation of cells and contact with anticancer agent) ↓ (7)Evaluation step (staining and fixing of cells, and analysis of image)

The following is a detailed explanation about each item of the flowsheet of Table 1.

[Test Materials]:

As the test materials, biopsy materials are taken out of human cancerpatients' tissue of breast cancer, lung cancer; colon cancer, andpancreatic cancer by a low invasive sampling method.

[Separating and Dispersing Treatments]:

Immediately after the sampling, the biopsy materials (biopsy cells).aresuspended into the above culture medium and then subjected to thebelow-mentioned separating and dispersing treatments. Incidentally, inthese treatments, the above culture medium is fitly used.

In the following way, the separating and dispersing treatments of cells((1) to (8)) are carried out according to states A) to C) of tissuelumps of the biopsy materials (collected biopsy tumor tissue). Thesetreatments enable reducing the loss in number of cells when collectingcells prior to the preliminary culture step.

(States of Tissue Lumps):

A) In the case where the tissue lumps are not smaller than 3 mm square,mechanical separation and enzymatic dispersion are needed, thusperforming the treatments (1) to (8) below.

B) In the case where the tissue lumps are 0.5 to 3 mm square (but notincluding 3 mm), mechanical separation is unneeded, and enzymaticdispersion is needed, thus performing the treatments (1) and (4) to (8)below.

C) In the case where the tissue lumps are smaller than 0.5 mm square,both mechanical separation and enzymatic dispersion are unneeded, thusperforming either the treatments (1) and (7) to (8) below or only thetreatment (8) below.

(Separating and Dispersing treatments of Cells):

(1) After the sampling, the biopsy materials are collected into acentrifugal tube of 15 ml containing 12 ml of the aforementioned culturemedium, and then centrifugal separation (1,400 rpm, 3 minutes) isperformed, and then the precipitated tumor tissue is collected.

(2) The centrifugally collected tumor tissue is suspended into 5 ml ofthe culture medium and then moved onto a dish of 6 cm.

(3) Only solid tissue is separately moved onto a dish of 10 cm and thenminced with a razor so as to be pasty, and solids are beforehandseparated so that the enzymatic treatment can rapidly be carried out,and then the residue is collected into a centrifugal tube of 15 ml, andthen centrifugal separation is performed at 1,400 rpm, for 3 minutes.Incidentally, in the case where many isolated cells are observed in theculture medium as used in the above treatment (2), they are separatelycollected into a centrifugal tube and then subjected to the preliminaryculture step directly without performing any enzymatic treatment.

(4) A 0.1% enzymatic treatment solution is added to the centrifugallycollected precipitate, and then they are shaken at 37° C. with a mildmixer for 30 to 60 minutes to carry out enzymatic dispersion of cells(digestion of intercellular interstitial tissue). Incidentally, thestate of the dispersion is fitly observed with a microscope. The shakingtime is set properly every original organ. Incidentally, the 0.1%enzymatic treatment solution was prepared by diluting a multienzymecomplex, containing collagenase (product of Nitta Gelatin Inc.),hyaluronidase (product of Sigma Co.), dispase (product of Godo ShuseiCo.), elastase (product of Wako Pure Chemical Industries, Ltd.) andDNase (product of. Sigma Co.), to 0.1% with the DF culture mediumsolution.

(5) Immediately after the enzymatic treatment, the volume is increasedwith the culture medium to stop the enzymatic reaction, and thencentrifugal collection is performed at 1,400 rpm, for 3 minutes.

(6) An amount of 1 to 2 ml of EGTA-Trypsin is added to the centrifugallycollected precipitate to perform the treatment for 3 to 5 minutes,thereby dispersing cells.

(7) Cells suspending is strongly carried out with the culture medium tomechanically loosen cells. (In the case where the state of the tissuelumps is A) above, a separating operation is carried out with a nylonmesh filter (φ300 μm).) Then, centrifugal separation is performed at1,400 rpm, for 3 minutes, and further, a density gradient centrifugationmethod is used to remove blood cell components and a portion offibroblast cells.

(8) Cells in the centrifugally collected precipitate are seeded onto abeforehand prepared culture supporting base and then subjected to thepreliminary culture step with the above culture medium.

[Preliminary Culture Step]:

A tubular plastic container having a surface area of 5.5 cm² was used asa supporting base in the preliminary culture step wherein a type-Icollagen gel (or type-IV collagen gel) had been coated as anextracellular matrix onto a surface of the container before the use. Thedispersion containing various cancer cells (including such as live tumorcells, fibroblast cells, dead tumor cells, dead tumor cell lumps, andenzymatically undigested substances), as obtained by the abovedispersing and separating treatments, was seeded onto the collagen gel,and then the culture was performed with the above culture medium in a 5%CO₂ incubator of 37° C.

When the dispersion solution containing such as cancer cells waspreliminarily cultured in the above way, it was seen that only the livecells adhered to and spread on the surface of the supporting base.Specifically, when microscopic observation was performed just after theculture and 24 hours later than the culture, it was seen that: the livecells adhered to and spread on the collagen gel, while the cancer-deadcells, the cancer-dead cell clumps, and the enzymatically undigestedsubstances were floating without adhering to the collagen gel.Thereafter, the culture medium was sucked, and the surface of thesupporting base was washed. When microscopic observation was performedin this state, the cancer-dead cells, the cancer-dead cell clumps, andthe enzymatically undigested substances were removed, and there wereseen only the live cells which had adhered to and spread on the collagengel.

[Cells-Seeding Step]:

The live cells which had adhered to and spread on the collagen gel weredetached by collagenase treatment, and then such as live tumor cellswere collected by centrifugal separation.

Incidentally, in the above preliminary culture, the cells were detachedfrom the supporting base basically after 24 hours and then collected,but in the case where only a small number of live tumor cells adheredalive to the surface of the supporting base, when the preliminaryculture time was extended (e.g. to about 2-7 days) to perform theculture the live tumor cells could sufficiently be proliferated in thestage of the preliminary culture. In other words, even if only a smallnumber of live tumor cells were obtained, the live tumor cells could beproliferated by the preliminary culture and then transferred to thesubsequent step. Incidentally, in the case where there is a possibilityof the extension of time, specifically, in the case where the culture isperformed for a long time of not less than 3 days, the type-I collagengel is preferable to the type-IV collagen gel as the extracellularmatrix being coated onto the surface of the supporting base.

[Embedding Step]:

The live tumor cells as obtained by the above-mentioned preliminaryculture and collection steps were embedded into a globular collagen geland cultured.

The embedded culture was carried out as follows: 1 volume of 10-foldconcentrated Ham's F12 culture medium (sodium bicarbonate-free) and 1volume of a reconstituting buffer solution (50 mM NaOH solutioncontaining 260 mM sodium bicarbonate and 200 MM-HEPES) were added to 8volumes of Cellmatrix Type-CD (0.3% acid-soluble Type-I collagensolution: product of Nitta Gelatin Inc.); and then the cancer cells asobtained in the aforementioned preliminary culture and collection stepswere added and well mixed into the resultant mixture; and then the finalconcentration was adjusted to 1×10⁵ cells/ml; and then the resultantmixture was preserved in ice.

Next, this collagen mixture solution was placed in hemispherical formonto 3 droplets of a 6-well multiplate at a rate of 30 μl/well using amicropipette and gelled to prepare a globular collagen gel.

A 3.0-ml portion of the culture medium containing anticancer agents wassuperposed on the globular collagen gel in which the cancer cells wereembedded and which was obtained in the embedding step, and then thecancer cells were contacted with the anticancer agents by keeping themin a 5% CO₂ incubator of 37° C. for 24 hours. Used as the anticanceragents were such as Taxol, Taxotere, Epi (Epirubicin), NVB (Navelbine),MMC (mitomycin C), CDDP (cisplatin), VDS (vindesine), VP-16 (etoposide),5-FU (5-fluorouracil) and ADM (adriamycin).

Then, the culture medium containing the anticancer agents was removed bysuction, and then 3.0 ml of an anticancer agent-free culture medium wasinstead added, and then the resultant mixture was shaken in a 5% CO₂incubator, thus washing the globular collagen gel. This procedure wasrepeated at least every 15 minutes for a total of 3 times, whereby theanticancer agents were removed from the globular collagen gel.

[Proliferative Culture Step]:

After the above agent contacting and removing steps, 4 ml of a culturemedium was added, and then the culture was effected in a 5% CO₂incubator of 37° C. for 7 days. The culture medium was exchanged as theneed arose. The above culture medium was used as the aforementionedculture medium.

[Evaluation Step]:

The neutral red (NR) stain method was used. In detail, the culturemedium was exchanged with a culture medium to which an NR-staining agenthad been added (NR concentration: 25-50 μg/ml), and then shaking waseffected for 2 hours in a 5% CO₂ incubator of 37° C., thus incorporatingthe NR into the cells.

After the treatment by the NR stain method, the culture mediumcontaining the NR was exchanged with PBS (Phosphate Buffered Saline),and then the sample was allowed to stand stationary in a room for 10minutes, thus removing the NR remaining outside of the cells.

Next, the PBS was exchanged with neutral buffered 10% formalin, and thenthe sample was allowed to stand stationary in a room for 40 minutes,thus fixing the cells and the NR as incorporated into the cells.

The globular collagen gel to which the cells had been fixed was immersedin distilled water for 10 minutes, thus removing the salts. The globularcollagen gel which had been displaced with the distilled water wasair-dried at room temperature, whereby a flat dried product was formed.At this time, the cultured cells were fixed in the dried collagen gelwhile containing the NR. These fixed cells exhibit no discoloration andcan endure even long-term preservation. The fixed cells were subjectedto an image-analyzing treatment. Incidentally, for this image-analyzingtreatment, there was used an image-analyzing method as disclosed inJP-A-115612/1998 (method for quantitative measurement of cancer cells).TABLE 2 Test materials Number of Primary culture Sensitivity test Typeof perform- Success Success Success Success cancer ances number ratenumber rate Example Breast 50 41 82.0% 28 68.3% cancer Lung 25 14 56.0%12 85.7% cancer Colon 10 8 80.0% 5 62.5% cancer Pancreatic 15 8 53.3% 675.0% cancer Total 100 71 71.0% 51 71.8% Compar- Breast 35 23 65.7% 1356.5% ative cancer Example Lung 18 6 33.3% 3 50.0% cancer Colon 10 330.0% 1 33.3% cancer Total 63 32 50.8% 17 46.6%

TABLE 3 Success in Test primary culture materials Success in Failure inFailure in Type of Number of sensitivity sensitivity primary culturecancer performances test test (LGR) NVC Contami. Example Breast 50 28 139 0 cancer Lung 25 12 2 11 0 cancer Colon 10 5 3 2 0 cancer Pancreatic15 6 2 7 0 cancer Total 100 51 20 29 0 Comparative Breast 35 13 10 11 1Example cancer Lung 18 3 3 11 1 cancer Colon 10 1 2 0 7 cancer Total 6317 15 22 9Notes):LGR: Low-growth-rateNVC: Non-viable-cellsContami.: Contamination<<Antibacterial Test for Culture Medium>>

The following antibacterial test was carried out so as to additionallyconfirm the killing action (bactericidal action) and themultiplication-inhibition action (bacteriostatic action) on the bacteriain the culture medium as used in Example 1.

The antibacterial test for the aforementioned culture medium of Example1 was carried out (where, as to the antibiotic agent concentration inthe culture medium, the test is carried out after the following dilutionseries are prepared).

As to the antibacterial test, the bactericidal action and thebacteriostatic action on the bacteria were measured. The kind of theobjective bacteria of the test was the following 6 bacteria in total:S.aureus IFO-12732, E. faecalis IFO-12970, and E. coli IFO-3301, whichare aerobic bacteria; C. albicans IFO-1385 which is true fungus, and B.fragilis ATCC-25285 and P. anaerobius ATCC-27337, which are anaerobicbacteria. Hereinafter, a M[C measurement method of confirming thebacteriostatic action and a MBC measurement method of confirming thebactericidal action are explained.

(MIC Measurement Method):

As to the aerobic bacteria and the true fungus (the aforementioned fourbacteria), the measurement was carried out according to procedures asdefined in the National Committee for Clinical Laboratory Standards(NCCLS), except that: in the method for diluting liquid culture mediumas defined in the Standards, the basal medium as mentioned in Example 1was used as culture medium for measurement; the culture temperature andtime are adjusted to 35° C. and 24 hours in the case of the aerobicbacteria, or adjusted to 25° C. and 72 hours in the case of the truefungus; the composition of the antibiotic agent (the kind of theantibiotic agent) was adjusted in the same way as of Example 1; and, asto the antibiotic agent concentration, the twice dilution series wereprepared, in which the antibiotic agent concentration (penicillin: 1mg/ml, kanamycin: 0.5 mg/ml, fungizone: 2.5 μg/ml, and vancomycin: 2mg/ml) in the culture medium of Example 1 was regarded as aconcentration of the original solution.

As to the anaerobic bacteria (the aforementioned two bacteria), themeasurement was carried out according to procedures as defined in thestandard method of the Chemotherapeutic Society of Japan, except that:in the method for diluting flat agar plate as defined in the Standards,agar culture medium (in a petri dish having a diameter of 9 cm)comprising the culture medium as mentioned in Example 1 was used asculture medium for measurement; the measurement environment was under anatmosphere of 5% CO₂, and the culture time was adjusted to 24 hours at35° C.; the composition of the antibiotic agent was adjusted in the sameway as of Example 1; and, as to the antibiotic agent concentration, thetwice dilution series were prepared by regarding the antibiotic agentconcentration of Example 1 as a concentration of the original solution.The anaerobic culture of the aforementioned condition was carried out inan anaerobic jar (Anaeropack KENKI, produced by Mitsubishi Gas ChemicalCo., Ltd.).

As to the respective concentration stages (respective dilution series)of the antibiotic agent in both the aforementioned two MIC measurementmethods, when the growth of bacteria was not observed with the naked eyeafter the culture, it was evaluated as (−). In reverse, when the growthof bacteria was observed, it was evaluated as (+). Their results wereshown in Table 4. Of the concentration stages that were evaluated as(−), the minimum antibiotic agent concentration is defined as MIC(minimum inhibitory concentration).

(MBC Measurement Method):

As to the aerobic bacteria and the true fungus (the aforementioned fourbacteria), one platinum loop with each of the culture mediums of all theconcentration stages evaluated as (−) after the culture in theaforementioned MIC measurement methods are transplanted toMueller-Hinton-Broth liquid culture medium which does not include anantibiotic agent each, but to which Ca²Mg²⁺ is added (bouillon formeasuring sensitivity, produced by Nissuw Co., Ltd.), and areaerobically cultured under the same conditions of the culturetemperature and time as the MIC measurement methods.

As to the anaerobic bacteria (the aforementioned two bacteria), anbactericidal absorbent cotton swab comes into contact with the agarculture medium of all the concentration stages evaluated as (−) afterthe culture in the aforementioned MIC measurement methods. Then, theagar culture medium is wiped out and transplanted to GAM agar culturemedium not including an antibiotic agent each, and then the culture isanaerobically carried out under an atmosphere of 5% CO₂ and under thesame conditions of the culture temperature and time as the MICmeasurement methods.

As to the respective culture stages in both the aforementioned two MBCmeasurement methods, when the growth of bacteria was not observed withthe naked eye after the culture, it was evaluated as (−). In reverse,when the growth of bacteria was observed, it was evaluated as (+). Theirresults were shown in Table 4. Of the stages that were evaluated as (−),the minimum oriented concentration (minimum antibiotic agentconcentration when the MIC is measured) is defined as MBC (minimumbactericidal concentration). TABLE 4 Anitbiotic agent twice dilutionseries 2¹ 2² 2³ 2⁴ 2⁵ 2⁶ 2⁷ 2⁸ 2⁹ 2¹⁰ 2¹¹ 2¹² 2¹³ (dilution) times timestimes times times times times times times times times times times S.aureus MIC (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (+) (+) IFO-12732MBC (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (+) (+) (+) E. faecalis MIC(−) (−) (−) (−) (−) (−) (−) (−) (−) (+) (+) (+) (+) IFO-12970 MBC (−)(−) (−) (−) (−) (+) (+) (+) (+) (+) (+) (+) (+) E. coli MIC (−) (−) (−)(−) (−) (−) (+) (+) (+) (+) (+) (+) (+) IFO-3301 MBC (−) (−) (−) (−) (−)(+) (+) (+) (+) (+) (+) (+) (+) C. albicans MIC (−) (−) (−) (−) (+) (+)(+) (+) (+) (+) (+) (+) (+) IFO-1385 MBC (−) (−) (−) (+) (+) (+) (+) (+)(+) (+) (+) (+) (+) B. fragilis MIC (−) (−) (−) (−) (−) (−) (+) (+) (+)(+) (+) (+) (+) ATCC-25285 MBC (−) (−) (−) (−) (−) (+) (+) (+) (+) (+)(+) (+) (+) P. anaerobius MIC (−) (−) (−) (−) (−) (−) (−) (−) (−) (−)(−) (−) (−) ATCC-27337 MBC (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (−)(+) (+)(Result and Consideration):

The culture medium as used in Example 1 displays a sufficient killingaction and multiplication-inhibition action on all kinds of theaforementioned objective bacteria, and these actions can be maintaineduntil the end of the culture even if the decrease of the potency may beexpected due to such as overlong culture.

<<Confirmation Test of Cell Toxicity According to Vancomycin Amount>>

The antibiotic agent in the culture medium as used in Example 1 includedvancomycin, but it was additionally confirmed whether the vancomycin hasa unfavorable influence upon characters of the aimed cells according tothe vancomycin amount or not.

Specifically, it is confirmed whether the vancomycin in the culturemedium of the preliminary culture step has a unfavorable influence uponthe cell proliferativity in the preliminary culture step, the cellproliferativity in the proliferative culture step, and the anticanceragent sensitivity test results or not, and then the following test wascarried out. Herein, the unfavorable influence means such an influenceas causes to decrease the cell proliferativity and success rate of theanticancer agent sensitivity test.

As to the test material and the number of cells as seeded (testmaterial/number of cells as seeded), the following were used:

-   C-1 (human colon cancer cell line)/1×10⁵ cells/well-   A-549 (human lung cancer cell line)/1×10⁵ cells/well-   MKN-28 human stomach cancer cell line)/1×10⁵ cells/well-   HFL-1 and WI-38 (human fibroblast cells line)/1×10⁵ cells/well-   Lung-k human primary lung cancer cells)/5×10⁵ cells/well (in the    preliminary culture step) or 2×10⁵ cells/well (in the proliferative    culture step)

The culture medium in the preliminary culture step was a culture mediumas used in Example 1, and only the concentrations of the vancomycinproduced by Wako Pure Chemicals Co., Ltd.) therein were settled in thefollowing conditions: 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0mg/ml, 2.5 mg/ml, 3.0 mg/ml, 4.0 mg/ml, and 5.0 mg/ml, and tests werecarried out in each condition. In addition, a sample to which thevancomycin was not added was regarded as control.

As the culture medium in the proliferative culture step, one (fortreatment of anticancer agents) obtained by adding the below-mentionedanticancer agents to the culture medium as used in Example 1 was used.In addition, the culture medium including no anticancer agent as used inExample 1 was used as control (for non-treatment of anticancer agents).

-   Taxol (0.05 μl/ml)-   Taxotere (0.1 μl/ml)-   Gemcitabine (1.0 μl/ml)-   Navelbine 0.05 μl/ml)    (Test Method in Preliminary Culture Step):

A culture supporting base was prepared by coating a 6-well multiplate(well surface area (basal surface area): 9.6 cm²) with collagen gel toform its layer. As to the five cell lines and one primary cell, theirseeding was carried out with the aforementioned number of cells asseeded, and the cells were overlaid with 5 ml of the culture medium.Thereafter, the monolayer culture was carried out for 48 hours.

After 24 hours from the beginning of the culture, the resultant livecells as adhered to the supporting base were collected. After the cellswere counted with a blood-cell counter board, the cell number per 1 mlof the culture medium was calculated, and the cell proliferativity inthe preliminary culture step was observed. Their results were shown inFIGS. 1 and 2.

(Test Method in Proliferative Culture Step):

The cells were collected from only the cells having a concentrationcondition in which the cell proliferativity was good in the preliminaryculture step, and the proliferative culture step was carried out by wayof the cells-seeding step and embedding step according to the specificprocedures the (1) to (3) below. Thereafter, the evaluation of the cellproliferativity and anticancer agent sensitivity test were carried out.

(1) The above five cell lines and one primary cell are mixed to acollagen solution so as to adjust to the aforementioned cells-seedingnumber, and then droplets (n=6) of 30 μl/droplet are prepared on a6-well test plate.

(2) After the droplets were gelled, the resultant droplets were overlaidwith the culture medium including the above various anticancer agents,and thereafter the culture is carried out for 7 days. Incidentally, theculture is carried out at 37° C. in a 5% CO₂ incubator.

(3) After 24 hours from the beginning of the culture, a dish (0 time) isstained and fixed.

(4) After 7 days from the beginning of the culture, the NR stain iscarried out, and the cells are fixed by neutral buffered formalin tocomplete the culture.

(5) The resultant test plate is quantitatively analyzed with an imageanalyzer, and then the unfavorable influence upon the cellproliferativity and the anticancer agent sensitivity test results(antitumor effect T/C (%): relative proliferation ratio T/C (%) betweena group non-treated with anticancer agents (C) and a group treated withanticancer agents (T), in the case where the ratio is not more than 50%,it is defined as having a sensitivity.) are observed. Incidentally, theevaluation of the cell proliferativity is carried out-on the basis ofthe value at 0 time. The results of the cell proliferativity in theproliferative culture step were shown in FIGS. 3 and 4, and the resultsof the anticancer agent sensitivity test were shown in FIGS. 5 to 10.

(Results and Consideration):

As to the unfavorable influence upon the cell proliferativity inproportion to the amount of the vancomycin as added in the preliminaryculture step, when the vancomycin was not added, the good cellproliferation was observed in the various cells after 48 hours from thebeginning of the culture. When the vancomycin was added, the cellproliferation in a condition of its concentration of not more than 2.0mg/ml was as good as that in a condition such that the vancomycin wasnot added. On the other hand, when the vancomycin was not added or thevancomycin concentration was at least not more than 3.0 mg/ml, the cellnumber when the cells-seeding step was carried out could be maintainedin the human primary culturing cells after 48 hours from the beginningof the culture, and the cell toxicity (cells-killing action) of theculture medium was not observed.

As to the unfavorable influence upon such as the cell proliferativity inthe proliferative culture step, used were the cells having good cellproliferativity after the preliminary culture step, namely the cellsthat were cultured in a condition such that the amount of the vancomycinas added was not more than 3.0 mg/ml. It was understood that these allhave good cell proliferativity after the 7 days from the culture, andthe unfavorable influence upon the cell proliferativity in theproliferative culture step is not found depending upon the amount of thevancomycin as added in the preliminary culture step. In addition, it wasunderstood that there is no unfavorable influence upon the anticanceragent sensitivity test results (antitumor effect T/C (%)) depending uponthe amount of the vancomycin as added in the preliminary culture step.

In addition, it is said that: according to the above additionalconfirmation (the confirmation of the unfavorable influence caused bythe amount of the vancomycin as added), the proliferating action thatthe culture medium had was displayed from the good cell proliferativity;in addition, the physiological activity-retaining action that theculture medium had was displayed from the anticancer agent sensitivitytest results.

COMPARATIVE EXAMPLE 1

The anticancer agent sensitivity test was carried out in the same way asof Example 1 except that: the culture medium as used was adjusted to thecomposition shown below; and the washing step was added between the step(1) and the step (2) as shown in the aforementioned flow sheet of Table1; and a culture flask having a surface area of 25 cm² was used as thesupporting base in the preliminary culture step. Then, the results wereevaluated. Incidentally, breast cancer cells, lung cancer cells, andcolon cancer cells were used as the cells subjected to the test inComparative Example 1. The results were shown in Tables 2 and 3.

As to the culture medium as optionally used in each step in ComparativeExample 1, used was a culture medium as prepared by adding penicillinand kanamycin to the following basal medium.

Basal Medium:

DF culture medium (DF: a mixed culture medium containing one volume ofDulbecco modified Eagle (DME) culture medium and one volume of Ham's F12culture medium)+10% FBS (FBS: fetal bovine serum)+5 μg/ml insulin(product of Sigma Co.)+10 ng/ml EGF (product of Collaborative Co.; EGF(epidermal growth factor))

Penicillin, kanamycin:

One 1 g-vial of penicillin (product of Toyama Chemical Co., Ltd.; forpenicillin injection) and 1 g potency of kanamycin (product of GIBCO BRLCo., Ltd.; sulfuric acid kanamycin reagent) are dissolved into 20 ml ofPBS, and the resultant solution is added to the basal medium in such anamount as gives a concentration of 0.2% (final concentration ofpenicillin and kanamycin 0.1 mg/ml each) on the basis of the basalmedium.

[Washing Step]:

Five sterilization-treated plastic dishes having diameters of 10 cm wereprepared, and about 25 ml of physiological saline for injection is addedto each dish. After biopsy materials as the test materials were sampled,they are picked out with a sterilized pincette and washed by moving themin the physiological saline in the first dish a few times. After thewashing in the first dish, the same procedure is also carried out forthe second to fifth dishes. Thereafter, the biopsy materials are movedin a culture medium as used in the above Comparative Example 1 to washthem, and then supplied to the separating and/or dispersing treatments.

Incidentally, the antibacterial test was carried out in the same way asof Example 1 so as to confirm the killing action (bactericidal action)and the multiplication-inhibition action (bacteriostatic action) on thebacteria in the culture medium as used in Comparative Example 1.

However, the kind of the objective bacteria of the test was thefollowing 4 bacteria in total: S.aureus IFO-12732, E. faecalisIFO-12970, and E. coli IFO-3301, which are aerobic bacteria; and C.albicans IFO-1385 which is true fungus, and the culture medium(comprising 0.1 mg/ml of penicillin and 0.1 mg/nl of kanamycin as theantibiotic agent) was the same as of Comparative Example 1. As to theantibiotic agent concentration, the twice dilution series were prepared,in which the antibiotic agent concentration in the culture medium ofComparative Example 1 was regarded as a concentration of the originalsolution. Then, the MIC measurement and MBC measurement were carried outin the same way as of Example 1. The results were shown in Table 5.TABLE 5 Anitbiotic agent twice dilution series 2¹ 2² 2³ 2⁴ 2⁵ 2⁶ 2⁷ 2⁸2⁹ 2¹⁰ 2¹¹ 2¹² 2¹³ (dilution) times times times times times times timestimes times times times times times S. aureus MIC (−) (−) (−) (+) (+)(+) (+) (+) (+) (+) (+) (+) (+) IFO-12732 MBC (−) (+) (+) (+) (+) (+)(+) (+) (+) (+) (+) (+) (+) E. faecalis MIC (−) (+) (+) (+) (+) (+) (+)(+) (+) (+) (+) (+) (+) IFO-12970 MBC (+) (+) (+) (+) (+) (+) (+) (+)(+) (+) (+) (+) (+) E. coli MIC (−) (−) (+) (+) (+) (+) (+) (+) (+) (+)(+) (+) (+) IFO-3301 MBC (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)(+) C. albicans MIC (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)IFO-1385 MBC (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)

EXAMPLE 2

<<Selection of Culture Supporting Base Area>>

As to the culture supporting base as used in the culturing processaccording to the present invention, especially the culture supportingbase usable in the preliminary culture step, the selection of thesurface area (when a container having the supporting base is a dish, thesurface area means a basal area.) was confirmed by experiment.

As the test materials, human primary cells (lung cancer cells) wereused. As to containers having the culture supporting base, 6-well,12-well, 24-well, and 48-well multiplates were used. The surface areas(base areas) of the wells that each plate has are 9.6 cm², 3.8 cm², 2.0cm², and 0.8 cm² respectively in order. The base surfaces of these fourdishes were coated with a Type-I collagen gel to form a basal layer,thus completing the culture supporting base.

In each dish, the cells having a cell number of 1×10⁵ to 1×10⁴cells/well (specifically, 4 patterns of 1×10⁵, 5×10⁴, 2×10⁴, and 1×10⁴cells/well) were seeded, and they were cultured at 37° C. in a 5% CO₂incubator. As to the culture medium, the culture medium as used inExample 1 was used.

After 24 hours from the beginning of the culture, the culture medium wasexchanged, and only the cells as fixed to the collagen gel werecollected. In addition, the values of the number of the live cells after24 hours and 72 hours from the beginning of the culture were evaluatedby colorimetric quantitative method. Then, the initial adhesion (OD₅₄₀)of the culture cells based on the surface area (base area) of theculture supporting base and the cell proliferativity ratio after 72hours from the beginning of the culture were calculated. The results ofthe initial adhesion and the results of the cell proliferativity ratiowere shown in FIGS. 11 and 12 respectively.

Incidentally, the above cell proliferativity ratio is defined as a ratioof the OD₅₄₀ after 72 hours from the beginning of the culture based onthe OD₅₄₀ after 24 hours from the beginning of the culture.

(Colorimetric Quantitative Method):

A MTT reagent (produced by Sigma Co.) was dissolved in a PBS so that theconcentration would be 5 mg/ml, and it was added to DF culture medium sothat the concentration would be 10%. A definite amount of this reagentcame into contact with the cells after the culture, and then was allowedto react for 3. to 4 hours. After the end of the reaction, the reagentwas removed, and a definite amount of DMSO (dimethylsulfoxide) was addedthereto, and then the resultant precipitated formazan crystal wasdissolved. (The resultant solution was colored blue.) The reactionliquid as obtained in this way was divided in a definite amount and putin a 96-well multiplate. Then, the colorimetric quantification wascarried out with a microplate reader (using a filter of 540 nm), andthen the OD of the live cells was measured.

(Results and Consideration):

From graphs as shown in FIGS. 11 and 12, it was understood that: whenthe supporting bases have surface areas (base areas) of 2.0 cm² and 3.8cm², the supporting bases have the most excellent initial adhesion and ahigh cell proliferativity ratio.

EXAMPLE 3

<<Selection of Culture Supporting Base Shape>>

As to the culture supporting base as used in the culturing processaccording to the present invention, especially the culture supportingbase usable in the preliminary culture step, the selection of thecontainer shape of the supporting base and its effect were confirmed byexperiment.

As the test materials, namely the biopsy cells, human primary cells(lung cancer cells and breast cancer cells) as obtained by sampling forbiopsy or by surgically resection were used. As to a container with theculture supporting base, the tube container as shown in FIGS. 13 and 14(the cutting face (flat face) is a portion where the cells adhere andthe culture is carried out, and the surface area of this portion is 5.5cm²) and a culture flask having a surface area of 5.5 cm² were used.Both containers are plastic containers. The collagen gel coating wasformed by overlaying the base of these two culture containers with a0.3% Type-I collagen solution (the same 0.3% Type-I neutralized collagensolution as used in the embedding step of Example 1) with a thickness of1 mm and gelling at 37° C., thus completing the culture supporting base.

After the above human primary cells (lung cancer cells and breast cancercells) were enzymatically dispersed for 1 to 2 hours, the unnecessarytissue was removed by a nylon mesh having a pore size of 300 μm, and thesize of the cell clump was adjusted. Next, the cell number was countedwith a hematocytometer, and a dispersion including 1×10⁵ cells wasprepared. Thereafter, the cells were seeded on the above various culturesupporting bases in two patters of 5×10⁴ cells and 1×10⁵ cells, and thenthey were cultured (preliminarily cultured) in a 5% CO₂ incubator of 37°C. In detail, in the above tube container and flask container each, 12samples having a cell number of 1×10⁵ cells and 8 samples having a cellnumber of 5×10⁴ cells of the lung cancer cells were seeded, and 4samples having a cell number of 1×10⁵ cells and 1 sample having a cellnumber of 5×10⁴ cells of the breast cancer cells were seeded, and thenthey were cultured. Incidentally, as to the culture medium, the culturemedium as used in Example 1 was used, and then it was overlaid with 10ml of the culture medium each.

After 48 hours from the beginning of the culture, the culture medium wasexchanged, and such as blood cell components and dead cells suspended inthe culture medium were removed, and the collagen gel layer wasdissolved by a 0.1% collagenase solution, and then only the cells asfixed to the collagen gel were collected.

The cells as collected from each supporting base were suspended in 0.5ml of a 0.3% Type-I collagen solution (the same 0.3% Type-I neutralizedcollagen solution as used in the embedding step of Example 1), and thena globular collagen gel (collagen gel droplet) of 30 μl was produced.Incidentally, 9 droplets were produced per sample, and the followinganalysis and measurement were carried out, and then their average valueswere defined as their results. After the above collagen gel dropletswere produced, the bioassay (colorimetric analysis) was immediatelycarried out by a Neutral Red staining liquid. Thereby, the imageabsorbency value is measured and the live cell number was converted fromthis absorbency. The graphs describing the results of measuring andcalculating the above absorbency (OD₅₄₀) and live cell number are shownin FIGS. 15, 16, 17, and 18.

Incidentally, FIG. 15 relates to the absorbency of the sample obtainedby seeding in a cell number of 1×10⁵ cells, FIG. 16 relates to the livecell number of the sample obtained by seeding in a cell number of 1×10⁵cells, FIG. 17 relates to the absorbency of the sample obtained byseeding in a cell number of 5×10⁴ cells, and FIG. 18 relates to the livecell number of the sample obtained by seeding in a cell number of 5×10⁴cells. In addition, as to the sample number in the figures, No. 4, 6,12, 13, and 23 relate to breast cancer cell samples, and the othersrelate to lung cancer cell samples. The broken line in FIGS. 16 and 18represents the number of the live cells as seeded.

In addition, for the purpose of evaluating the maintenance of cell lifeafter the preliminary culture, the collagen gel droplet in which thecells after the preliminary culture was embedded was prepared asmentioned above, and the embedded culture of the collagen gel dropletwas carried out for 7 days. Thereby, the cell proliferativity ratio forthe 7 days embedded culture was calculated, and the cell proliferativityof all the cell samples that were preliminarily cultured in the abovetube and flask was confirmed. Specifically, for each sample, the cellscollected from the individual supporting bases were suspended in a 0.3%Type-I collagen solution (the same 0.3% Type-I neutralized collagensolution as used in the embedding step of Example 1) so that theconcentration would be 1×10⁵ cells/ml, and 6 globular collagen gels.(collagen gel droplets) of 30 μl were produced per sample. As to thethree droplets of these, the bio-assay (colorimetric analysis) by aNeutral Red staining liquid was immediately carried out after thedroplets were prepared, and the image absorbencies were measured, andthen the average value was calculated. As to the residual threedroplets, the colorimetric analysis was carried out in the same way asshown above, and the average is calculated after the serum-free culturewas carried out for 7 days. The cell proliferativity ratio of eachsample was calculated as a ratio of the image absorbency value after 7days' embedded culture based on the image absorbency value before theculture.

As to the cell proliferativity ratio as calculated in each sample, thecell proliferativity ratio of the cells oriented from the preliminaryculture in the tube container was regarded as 100%, and the cellproliferativity ratio of the cells oriented from the preliminary culturein the flask container was calculated as a relative cell proliferativityratio (relative ratio (%)). The graphs showing the results of the aboverelative ratio (%) are shown in FIGS. 19 and 20. In FIGS. 19 and 20, thecell proliferativity ratio was not more than 0.8 (Low-growth-rate) andthe decrease of the cell viability was considerably observed in the dataof the flask container of the samples 10, 14, 15, 16, 20, 22, 24, and25, and in the data of the tube container of the samples 16 and 24.

Incidentally, FIG. 19 shows a relative ratio (%) as to the samplesobtained by seeding and then culturing in a cell number of 1×10⁵ cells,and FIG. 20 shows a relative ratio (%) as to the samples obtained byseeding in a cell number of 1×10⁵ cells. In addition, as to the samplenumber in the figures, No. 4, 6, 12, 13, and 23 relate to breast cancercell samples, and the others relate to lung cancer cell samples, in thesame way as mentioned above.

(Colorimetric Analysis by a Neutral Red Staining Liquid):

The Neutral Red bio-assay (colorimetric analysis) was carried out in thefollowing procedure.

The culture dish where the collagen gel droplet was prepared wasoverlaid with the culture medium as obtained by adding a Neutral Red(hereinafter, referred to as NR) staining agent (NR concentration of 25to 50 μg/ml), and they were stirred and cultured in a 5% CO₂ incubatorof 37° C. for 2 hours. Thereby, the NR was incorporated in the cells bya living action (phagocytosis) that the cell had. According to thisoperation, only the live cells can be stained and distinguished. Theculture medium containing the NR was washed and removed by a PBSPhosphate Buffered Saline), and the cells and the NR pigment asincorporated were fixed by a 10% neutral buffered formalin solution.Thereafter, a flat measuring sample was obtained by airdrying.

The above measuring sample was calorimetrically quantified with an imageanalyzer. Thereby, the integrated image concentration of only the livecancer cells as colored by NR staining was converted to an absorbencyvalue (OD₅₄₀) according to the Lambert-Beer's formula. In addition, thenumber of the live cancer cells in the globular collagen gel wasdetermined from the resultant absorbency value by converting in thefollowing way.

At first, when the cells were seeded so that the number of the livecells as included per collagen droplet (30 μl) would be 2,500 cells, itwas confirmed by a preliminary experiment that the image absorbencyvalue was 20 on average. Accordingly, the above number of the live cellswas calculated according to the following converting equation:Number of live cells piece/0.5 ml)=(Image absorbency value/20]×2,500(cell number)×16 (droplet number)

From the value as obtained according to the above converting equation,the yield of the live cells in the various culture supporting base wasevaluated.

(Results and Consideration):

In a graph as shown in FIG. 15, from the image absorbency values,observed was the significant difference (t-test=significant level 0.05)such that the tube container is superior because of the difference ofthe container (difference of the supporting base shape) in 11 samplesamong 16 samples.

In a graph as shown in FIG. 16, obtained was the result that: in almostall the samples of the tube container, the numbers of the live cellswere nearly as large as or greatly larger than the cell number when theseeding was carried out. However, in the case of using the flaskcontainer, in many samples, the numbers of the live cells were nearly aslarge as the cell number when the seeding was carried out, but in notless than half of the samples, the numbers of the live cells weresmaller than the cell number when the seeding was carried out.Accordingly, it was observed that the cell collection ratio was lowered.From the above fact, when the tube container is compared with the flaskcontainer, it was confirmed that there were many cases where the tubecontainer could achieve enough proliferation to greatly exceed the cellnumber when the seeding was carried out, and could particularlyfavorably satisfy the present invention object.

In a graph as shown in FIG. 17, from the image absorbency values,observed was the significant difference (t-test=significant level 0.05)such that the tube container is superior because of the difference ofthe container (difference of the supporting base shape) in 6 samplesamong 9 samples.

A graph as shown in FIG. 18 shows results in the case that the celldensity was lowered when the seeding was carried out. There were a fewcases where, in every supporting base, the numbers of the live cellsexceeds the cell number when the seeding was carried out, and there werea few samples in which the numbers of the live cells were nearly aslarge as the cell number when the seeding was carried out. However, whenthe tube container is compared with the flask container, it wasconfirmed that the tube container had a higher cell collection ratio ingeneral and the numbers of the live cells therein were more nearly aslarge as the cell number when the seeding was carried out.

In graphs as shown in FIG. 19 and 20, when the preliminary culture iscarried out using the tube container, it is found every sample hascomparatively high cell proliferativity, and culture tests such as ananticancer agent sensitivity test can sufficiently be carried out andcontinued.

Incidentally, as to 25 embedded and cultured samples as shown in FIGS.19 and 20, the samples in which the sufficient cell growth rate (notless than 0.8 time as large as the number of cells as seeded in theembedding culture) could not be obtained was: 8 samples when the flaskcontainer was used; and 2 samples when the tube container was used.

From the above test results, it was found that: in the case where theabove tube container as shown in FIGS. 13 and 14 was used as a containerwith the culture supporting base in the culture of the biopsy cancercells, not only the live cell collection ratio was improved when thepreliminary culture was carried out, but also very excellent effects asto maintenance of the cell proliferativity (cell proliferation ability)was displayed in the subsequent culture, in comparison with the casewhere the above flask container was used. Furthermore, the test successrate can be improved in such as an anticancer agent sensitivity test.

Various details of the invention may be changed without departing fromits spirit not its scope. Furthermore, the foregoing description of thepreferred embodiments according to the present invention is provided forthe purpose of illustration only, and not for the purpose of limitingthe invention as defined by the appended claims and their equivalents.

1-9. (canceled)
 10. A kit for culturing animal cells, which comprisesthe following constituent articles: a collagen solution; a concentratedculture medium; a reconstituting buffer solution; an enzyme fordispersing cells; a [tube] device for a culture supporting base; aculture medium for preliminary culture; a serum-free culture medium; anda cell-dyeing agent.
 11. A kit for culturing animal cells, comprising: abase having a culture surface of about 0.01 to 25.0 cm²; anextracellular matrix on the surface area of the base for enabling theanimal cells to adhere and proliferate; a culture medium having aphysiological activity action on the animal cells and a killing actionand/or multiplication inhibition action on bacteria in the preliminaryculturing of the animal cells; and a culture substrate as anextracellular matrix for secondary culture having a proliferatingaction.
 12. The kit according to claim 11, wherein the base is selectedfrom the group consisting of a tube, a plate, a microwell plate, amicrotiter plate, and flask.
 13. The kit according to claim 11, whereinthe culture substrate as an extracellular matrix for secondary cultureincludes a culture medium, a buffer, or mixture thereof.
 14. The kitaccording to claim 13, which further comprises a serum-free culturemedium.
 15. The kit according to claim 13, which further comprises acell dyeing agent.
 16. The kit according to claim 13, which furthercomprises a medicine.
 17. The kit according to claim 11, wherein theculture medium includes an antibiotic.
 18. The kit according to claim17, wherein the antibiotic is vancomycin in an amount of 0.01 to 3mg/ml.
 19. The kit according to claim 17, wherein the culture mediumfurther contains a cell growth factor, a serum, or mixture thereof. 20.The kit according to claim 11, which further comprises an enzyme fordispersing the animal cells in the culturing liquid.
 21. The kitaccording to claim 11, wherein the extracellular matrix is selected fromthe group consisting of collagen, fibronectin, laminin, vitronectin,cadherin, gelatin, peptides, and integrin.
 22. The kit according toclaim 17, wherein the antibiotic includes at least two members selectedfrom the group consisting of cell-wall-synthesis inhibitors,protein-synthesis inhibitors, nucleic-acid-synthesis inhibitors, andantifungal agents.